Flaviviruses such as West Nile (WN), Japanese encephalitis (JE), St. Louis encephalitis, and tick-borne encephalitis (TBEV) viruses are important neurotropic human pathogens, causing a devastating and often fatal neuroinfection. During the past two decades, both mosquito- and tick-borne flaviviruses have emerged in new geographic areas of the world where previously they had not been endemic and caused outbreaks of diseases in humans and domestic animals (TBEV in North Europe and Japan;JE in Australia and Oceania;Usutu virus, an African flavivirus, in Central Europe;WN in North and South America). Despite the availability of formalin-inactivated TBEV vaccines, there are more than 10,000 hospitalized TBE cases reported annually in endemic areas of Europe and Asia with mortality rate of up to 30%. An effective vaccine that induces a durable immunity against TBEV is urgently needed to protect humans since the geographic range and magnitude of TBEV infection continues to expand and increase. The inactivated TBEV vaccines produced in Europe are not licensed in the USA. The live attenuated TBEV vaccine candidates are being developed in the Neurotropic Flavivirus Section of the LID using a strategy based on chimerization of a neurovirulent TBEV with a non-neuroinvasive, mosquito-borne dengue-4 virus (DEN4d30) that contains an attenuating mutation, a 30 nucleotide deletion in the 3non-coding region. Chimeric tick-borne encephalitis/dengue virus (TBEV/DEN4d30) that contains the structural protein genes of a highly virulent TBEV demonstrates moderate levels of immunogenicity and protective efficacy in mice and monkeys, but retains an unacceptably high level of neurovirulence based on the results of clinical observations and analysis of virus-induced histopathologoly in the CNS of monkeys. Therefore, further attenuation of TBEV/DEN4d30 neurovirulence was achieved by introducing amino acid substitutions that had previously been shown to reduce replication of tick-borne Langat virus (LGT) or DEN4 in suckling mouse brain. When three amino acid substitutions (Lys315 >Glu in the structural envelop E protein and Asp654Arg655 >AlaAla in the non-structural NS5 protein) were introduced into the TBEV/DEN4d30 genome, the resulting virus (TBEV/DEN4d30-E315-NS5-654,655) demonstrated the desired properties of an acceptable live attenuated vaccine candidate. In FY 2010, a seed TBEV/DEN4d30-E315-NS5-654,655 virus was generated, and we demonstrated that it was highly attenuated for neurovirulence, neuroinvasiveness, virus-induced histopathology and replication in the CNS of mice, and was unable to infect or replicate in Aedes aegypti mosquitoes and Ixodes scapularis ticks. The poor infectivity for both potential insect vectors reinforces the safety of this vaccine candidate for the environment and for use in humans. In addition, we found that the protective efficacies in mice immunized with a single dose of this vaccine candidate or with three doses of the commercial inactivated TBEV vaccine were similar when animals were challenged with wild-type Central European or Far Eastern strains of TBEV. Thus, TBEV/DEN4d30-E315-NS5-654,655 virus is a promising TBEV vaccine candidate, but its ability to induce a protective TBEV-specific immune response and its level of neurovirulence in the CNS of non-human primates need to be evaluated prior to testing in humans. A live attenuated WN/DEN4d30 virus vaccine is being developed in the LID to protect humans against WN disease. In FYs 2006-2009, in the clinical trials in healthy adult volunteers, the WN/DEN4d30 vaccine was well-tolerated, safe, and induced a potent and durable WN antibody response at an immunization dose of 1,000, 10,000, or 100,000 PFU. Further studies of neurovirulence in monkeys are necessary to provide the additional evidence of safety of this vaccine for the CNS of non-human primates prior to its use in the risk group of volunteers >50 years of age. In FY 2010, a comparative analysis of neurovirulence and neuropathogenesis of the WN/DEN4d30 vaccine versus wild type WN virus and yellow fever (YF) 17D vaccine in the CNS of non-human primates was initiated and will be evaluated in a manner similar to that performed for TBEV/DEN4d30. Since neurotropic flaviviruses evoked a strong cellular inflammatory response and neurodegeneration in the CNS of monkeys, we have developed a high-throughput automated image analysis for the quantitative evaluation of the CNS infiltration with peripheral immune cells (CD3, CD4, CD8, and CD20 lymphocytes) and the response of CNS resident cells (microglial activation and neuronal degeneration). We found that quantitative estimates of immunoreactivity for CD3 T cells and CD20 B cells correlated remarkably well with results of traditional semi-quantitative histopathological scores for cellular inflammatory infiltration in the CNS of monkeys inoculated with YF 17D, LGT or TBEV/DEN4d30 attenuated viruses. In addition, quantitative analysis of immunoreactivity for neuron-specific antigen NeuN is a useful and reliable method for the assessment of neurodegeneration and neuronal loss. Our data indicate that a high CD4:CD8 T cell ratio in the CNS inflammatory infiltrates induced in response to YF 17D infection is a major factor that differentiates this successful vaccine from a more neurovirulent LGT or TBEV/DEN4d30 virus. It is likely that the balanced response of T and B cells within the CNS of monkeys induced with YF 17D vaccine virus plays an important role in the recovery from CNS infection and might serve as a reference to evaluate the safety of new live flavivirus vaccine candidates. In order to develop a general strategy for the rational design of safe and effective live flavivirus vaccines, we have initiated a project, in which we explored the ability of the brain tissue-expressed cellular microRNAs to control the neurotropism of flaviviruses, carrying complementary microRNA-target sequences. We anticipate that these viruses will replicate in peripheral non-CNS tissues and induce a strong adaptive immune response, but will be restricted in their ability to replicate in the CNS, since the CNS-expressed microRNAs will recognize the introduced complementary target sequences in the viral RNA genome and limit its translation, replication, and assembly into a virion. As a model virus for modification, we selected a chimeric TBEV/DEN4 that contained the structural protein genes of a highly virulent TBEV. The inclusion of just a single copy of the target for a brain-enriched mir-9, mir-124, mir-128, mir-218, or let-7c microRNA into the TBEV/DEN4 genome was sufficient to completely prevent the development of lethal encephalitis in mice infected directly in the brain with a large dose of virus. Viruses bearing a complementary target for mir-9 or mir-124 were highly restricted in replication in primary neuronal cells and had the limited access into the CNS of immunodeficient mice, but retained the ability to induce a potent humoral immune response in monkeys. This work suggests that a microRNA-targeting approach to control the virus tissue tropism could provide a new basis for future design of safe and effective live virus vaccines against neurotropic flaviviruses.